Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions
- Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions
- 황나래; 임승희; 김영미; 정재호; 송은주; 이윤지; 이진희; 최선; 이공주
- active site cysteine; cysteic acid; glyceralde-hyde-3-phosphate dehydrogenase; GAPDH; oxidative stress; 54 kDa nuclear RNA-binding protein (p54nrb); topoisomerase
- Issue Date
- The Biochemical journal
- VOL 423, NO 2, 253-264
- Knowledge of the cellular targets of ROS (reactive oxygen
species) and their regulation is an essential prerequisite for understanding
ROS-mediated signalling. GAPDH (glyceraldehyde-3-
phosphate dehydrogenase) is known as a major target protein
in oxidative stresses and becomes thiolated in its active site.
However, the molecular and functional changes of oxidized
GAPDH, the inactive form, have not yet been characterized.
To examine the modifications of GAPDH under oxidative
stress, we separated the oxidation products by two-dimensional
gel electrophoresis and identified them using nanoLC-ESI-q-
TOF MS/MS (nano column liquid chromatography coupled to
electrospray ionization quadrupole time-of-flight tandem MS).
Intracellular GAPDH subjected to oxidative stress separated into
multiple acidic spots on two-dimensional gel electrophoresis
and were identified as cysteine disulfide and cysteic acids on
Cys152 in the active site. We identified the interacting proteins
of oxidized inactive GAPDH as p54nrb (54 kDa nuclear RNAbinding
protein) and PSF (polypyrimidine tract-binding proteinassociated
splicing factor), both of which are known to exist as
heterodimers and bind to RNA and DNA. Interaction between
oxidized GAPDH and p54nrb was abolished upon expression of
the GAPDH active site mutant C152S. The C-terminal of p54nrb
binds to GAPDH in the cytosol in amanner dependent on the dose
of hydrogen peroxide. The GAPDH–p54nrb complex enhances
the intrinsic topoisomerase I activation by p54nrb–PSF binding.
These results suggest that GAPDH exerts other functions beyond
glycolysis, and that oxidatively modified GAPDH regulates its
cellular functions by changing its interacting proteins, i.e. the
RNA splicing by interacting with the p54nrb–PSF complex.
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